S. aureus activates TNF signaling pathways PROJECT SUMMARY Staphylococcus aureus remains a major cause of human infection and the prevalence of invasive staphylococcal disease has increased in the past few years. Protein A or SpA is a highly conserved surface component of S. aureus that has multiple interactions with the immune system. Discrete domains of SpA activate TNFa signaling, EGFR-mediating responses and the activation of the type I interferon cascade. The SpA IgG binding domain activates TNFR1, an interaction that is central to the pathogenesis of pneumonia. We postulate that the SpA-TNFRI interaction is critical in staphylococcal invasion across mucosal barriers through its activation of RhoA GTPases that perturb actomyosin dynamics and disrupt epithelial tight junctions. The SpA IgG binding domain also is an EGFR ligand, stimulating its phosphorylation and activating the ADAM17 metallo-protease. We predict that EGFR also mediates the internalization of SpA, which, through its Xr domain, activates the type I interferon cascade, inducing epithelial production of IFN-b and Jak-STAT phosphorylation in a TLR4/TRIF-dependent fashion. Staphylococcal activation of type I interferons via the TLR4 cascade is postulated to have a major role in the pathogenesis of pneumonia and sepsis. As the Xr domain is a common site of in vivo mutation, duplication and deletion, we will determine if these mutations selected in vivo are generated specifically in response to immune pressure and establish how polymorphisms in Xr contribute to the virulence of S. aureus.